Monoclonal antibodies against dengue viruses and other flaviviruses, including the tick-borne encephalitis virus, Japanese encephalitis and West Nile virus, are valuable for analysis of the virus structure, replication, pathogenesis and possible use for prevention of infections by these viruses. The four dengue virus serotypes are most important in terms of human morbidity and geographic distribution. Several live-attenuated dengue vaccine formulations are currently in clinical trials. However, the safety of such dengue vaccines remains a concern. Passive immunization using monoclonal antibodies from humans or non-human primates represents an attractive alternative to a dengue vaccine. Repertoire cloning was performed to recover Fab antibody fragments from bone marrow mRNA of a chimpanzee infected with all four dengue virus serotypes. Earlier, we recovered Fab antibodies highly efficient for neutralization of dengue type 4 virus following panning with dengue type 4 virus. In order to recover Fab antibodies against the other three dengue virus serotypes, repertoire cloning of the same phage library was also performed using dengue type 1, 2 and 3 viruses as panning antigens. Instead of a type-specific antibody, dengue virus-neutralizing Fabs that were cross-reactive to all dengue serotypes were identified. One of the neutralizing Fabs was selected to construct a full-length IgG1 antibody in combination with the human sequences. The humanized antibody proved to be efficient for cross-neutralization of dengue type 1 and type 2 viruses. This antibody also neutralized dengue type 3, type 4 viruses and West Nile virus, but a slightly reduced titer. To gain an insight into the antigenic specificity and the mechanisms of neutralization, we have analyzed the epitope determinants of the chimpanzee Fab antibodies, which had been shown to be dengue type specific or broadly reactive to flaviviruses. Sequence analysis showed that one antigenic variant contained a Gly-to-Val substitution at position 106 within the flavivirus-conserved fusion peptide loop of E and another variant contained a His-to-Gln substitution at position 317 in E. Both substitutions lowered the pH threshold for membrane fusion. In the three-dimensional structure of E Gly106 in domain II and His317 in domain III of the opposite E monomer were spatially close. Similar analysis showed that Fab 1A5 appears to recognize a novel epitope that has not been mapped before with a flavivirus monoclonal antibody. As an extension of these studies, a panel of chimpanzee Fabs that neutralized the Japanese encephalitis virus have been recovered and characterized and recently humanized. These humanized monoclonal antibodies represent attractive candidates for further development of a passive immunization strategy against dengue and other flaviviruses-associated diseases.